Document ppnMjNY2DjO9vVdGM1njy06Rk

A R 226 'C o ' Oral Teratology Study of FC-95 in Rats Experiment No.: Conducted At: Dosing Period: Study Director: 0680TR0008 Safety Evaluation Laboratory Riker Laboratories, Inc. St. Paul, Minnesota July 14, 1980 through July 24, 1980 E. G. Gortner E. G. Gortner Date Senior Research Technologist Animal Reproduction-Teratology Study Director f >\ -1% ^ ^ 1^ E. G. Lamprecht Research Veterinary Pathologist c Date .Y __ /2/sjf/?6 M. T. Case, DVM, PhD Date Manager, Pathology-Toxicology Safety Evaluation Laboratory 000615 1 Summary Oral administration of FC-95 at doses of 10, 5 and 1 mg/kg/day to pregnant Sprague-Dawley rats during days 6 through 15 of gestation (period of organogenesis) resulted in fetuses with teratogenic changes in the lens of the eye. The teratogenic effect was a developmental eye abnormality which appeared to be an arrest in development of the primary lens fibers forming the embryonal lens nucleus, followed by secondary aberrations of the secondary lens fibers of the fetal nucleus. The lens abnormality occurred in all FC-95 dose groups, but the proportion of fetuses with the lens changes was significantly higher them the control group only in the 10 mg/kg/day group. FC-95 administration was maternally toxic only to the 10 mgA g / d a y group. At gestation days 12 through 20 their mean maternal body weights were significantly lower than the controls. FC-95 was not maternally toxic to the 5 and 1 mg/kg/day groups. FC-95 was not embryotoxic and did not affect the ovaries or reproductive tract contents of the dams. The compound did not produce an increase in the number or proportion of abnormal fetal skeleton aberrations. 000G16 2 Introduction This teratology study^ of FC-95 in rats was conducted to evaluate the embryotoxic and teratogenic effects of orally administered FC-95. The study was sponsored by 3M Commerical Chemical Division, St. Paul, Minnesota and was conducted by the Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. The compound administration period was from July 14 through July 24, 1980. The protocol and list of the principal participants and supervisory personnel can be found in Appendices I and II respectively. All portions of this study were conducted according to the Good Laboratory Practice (GLP) regulations and the Safety Evaluation Laboratory Standard Operating Procedures (see Appendix III for Quality Assurance Unit statement). The storage location for specimens, raw data and a copy of the final report is maintained in the Safety Evaluation Laboratory's record archives. Methods Time mated Spraque-Dawley derived rats were obtained from Charles River Breeding Laboratory and assigned cages according to a computer-generated random numbers table. The rats were then divided into four groups of 22 animals weighing 175 to 261 grams. The rats were housed individually in hanging stainless steel cages with^wire mesh floors and fronts in a temperature and humidity controlled room. Food-- and water were available ad libitum. The lights were on a 12 hour light/dark cycle. The animals were observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights were recorded on days 3, 6, 9, 12, 15 and 20 of gestation and the rats dosed accordingly using a constant dose volume of 5 ml/kg of body weight. The four groups were dosed with FC-95 (Lot 640) suspended daily in corn oil at 0, io, 5 or 1 mg/kg/day. FC-95 was administered daily by oral intubation with a syringe equipped with a ball-tipped intubation needle to the rats on days 6 through 15 of gestation (day 0 indicated by sperm-positive vaginal smear). FC-95 analytical characterization (see Appendix IV) was provided by 3M Commercial Chemical Division, St. Paul, Minnesota. All animals were sacrificed on day 20 by cervical dislocation and the ovaries and uterus, including its contents, were examined immediately to determine the following: number of corpora lutea, number of viable fetuses, number of resorp tion sites, pup weights and sex, and any gross fetal abnormalities. Approximately one-third of the fetuses were fixed in Bouin's solution for subsequent free-hand sectioning by the Wilson technique to determine visceral abnormalities. The remaining fetuses were preserved in alcohol for clearing and staining of the skeleton with alizarin red to detect skeletal abnormalities. Selected free-hand sections were processed for histological evaluation. g- Riker Experiment No. 0680TR0008 -- Purina Laboratory Chow, Ralston Purina Company, St. Louis, MO 000617 4 3 Results FC-95 administered during the period of organogenesis was toxic to the high dose group (10 mg/kg/day) maternal rats. The mean body weights of all dose groups were similar at gestation days three through nine (Table 1, Appendix V ) . At gestation days 12 through 20 the high dose group rats weighed significantly less than controls (0 mg/kg/day). The mean maternal bodyweights of mid (5 mg/kg/day) and low (1 mg/kg/day) dose groups were not different from the controls throughout the study. Even though FC-95 was maternally toxic at the high dose level, no compound-related clinical signs were observed in any of the dose groups. FC-95 was not embryotoxic and did not affect the ovaries or reproductive tract contents of the dams. The mean number of male, female, total and dead fetuses; the mean number of resorption sites, implantation sites, corpora lutea and mean fetus weights of the three FC-95 dose groups were not significantly different from the controls (Table 2, Appendix VI). The high dose group did have a lower mean number of viable male, female and total fetuses than the other three groups which resulted from a lower number of embryos at the start of the study. Con tributing pieces of evidence to the lower number of high dose embryos are the low mean number of implantation sites, corpora lutea, resorption sites and the absence of dead fetuses. FC-95 did not cause compound-related abnormal gross fetal findings (Table 3), nor did FC-95 treatment produce an increase in the number or proportion of abnormal fetal skeletal aberrations. Fetal skeleton results of the three compound treated groups were not significantly different from the control group (Table 4) . The incidence and proportions of stemebrae nonossified and associated changes of sternebrae assymetrical, stemebrae bipartite and one stemebrae missing were unusually high in all dose groups of this study including the control group. FC-95 was teratogenic in the rat at all dose levels administered in this study. The teratogenic effect was a developmental eye abnormality which appeared to be an arrest in development of the primary lens fibers forming the embryonal lens nucleus, followed by secondary aberrations of the secondary lens fibers of the fetal nucleus. All eye abnormalities were localized to the area of the embryonal lens nucleus although a variety of morphological appearances were present within that location. The range of morphological appearances as (observed under the dissecting microscope varied from a slight discoloration of the lens near the anterior margin to a dark colored oval area, often containing a cleft, extending from beneath the lens epithelium to half-way through the lens posteriorly. Histologically the discolorations were due to presence of lens vesicle remnants surrounding the abnormal embryonal lens nucleus. One of the most severly affected eyes had most of the embryonal lens nucleus replaced by sinus spaces containing red blood cells. Also contributing to the discolorations were primary lens fibers which appeared to have not elongated. These lens fibers were tortuous and lacked nuclei in a normal lens bow of nuclei. Secondary lens fiber development progressed normally except immediately surrounding the abnormal embryonal nucleus. Secondary aberrations of secondary lens fibers included the bending of the fibers around the abnormal oval area, the subsequent formation of prominant anterior and posterior Y sutures of the converging fibers and lens vesicle remnants surrounding the embryonal nucleus. 000618 4 The lens abnormality occurred in all dose groups except the control group. The proportion of fetuses with the lens abnormality in one or both lenses was significantly higher in the high dose group than the control (Table 5). The lens abnormality recorded for one control fetus under the dissecting microscope was an artifact when evaluated by transmission light microscopy. A no-effect dose level for the teratogenic lens abnormality was not established in this study. Discussion Optimal visual functional requirements are met during embryonic development by the differentiation of highly specialized populations of cells from undifferentiated precoursers and by the coordinated morphogenesis of the resulting tissues. Both processes are controlled to a remarkable extent by interactions which occur among emerging tissues. Each tissue of the eye is brought to its final state of differentiation, its cell population size and its definitive geometry, not only by intrinsic processes, but also by extrinsic influences exerted by neighboring tissues^-. The embryonal origin of the lens is undifferentiated ectoderm. The tip of the optic vesicle, presumably the neural retina, plays the final role in inducing lens from overlying ectoderm and in aligning the lens precisely with the rest of the eye. Alternative or sequential action of tissues derived from endoderm (foregut) and mesoderm (heart) on the same target tissue decreases the probability that lens formation would be aborted by accidents during the early phases of induction. While the nature of the inductive influence remains unknown, there are indications that substances may be transferred from the presumptive neural retina to the overlying ectoderm during induction. A prolonged period of inductive interaction not only increases the probabililty that lens induction will occur successfully in the face of interference, but provides a mechanism for continuously adjusting the size, shape, position and orientation of the lens to that of the retina2 . During the early stages of the inductive process, the ectodermal cells immediately overlying the tip of the optic vesicle elongate prependicularly to the body surface to form a thickened disc (lens placode). The change in cell shape is accomplished without change in cell volume. The number of cells, however, continues to increase during this period. Toward the end of lens placode formation, acidophilic fibrils appear in the apices of the lens placode cells. At about this time, the placode invaginates to form the lens cup. This invagination is independent of the concomitant invagination of the underlying optic vesicle, and is probably due to forces operating within the lens ectoderm. As the lens cup deepens, its opening (lens pore) becomes progressively constricted until its lips meet and fuse, cutting off the lens vesicle internally and re-establishing continuity in the overlying ectoderm. Closure of the lens pore is attended by, and possibly accomplished by, a local and temporaty restricted wave of cell death. Following closure of the lens pore, the cells at the back of the lens vesicle continue to elongate, under the influence of the neural retina, to form the lens fibers. As the fibers grow the cavity of the lens vesicle is obliterated. The lens cells toward the ectoderm, which do not elongate further, form the lens epithelium2 . 000619 5 The cuboidal lens epithelial cells which face the cornea continue to grow after the lens vesicle forms. As the cells rotate through the equator region, they take their places on the surface of the growing fiber mass. These cells differ entiate into secondary lens fibers at the equator and elongate rapidly toward the poles of the lens where they meet with other fibers in planes of junction called sutures. As secondary fibers grow their nuclei become positioned at about the center of the fibers and form a convex lens bow outward. Since the newer fibers are always deposited superficially, the oldest fibers in the lens come to lie centrally and are referred to collectively as the lens nucleus. With time the lens cell nuclei in this region become pycnotic and finally disappear. The cell fibers, however, are not broken down and removed but remain in place. Thus the size and shape of the lens are controlled by factors which control the number, size and shape of lens cells2. The teratogenic effect of FC-95 probably occurred during the portion of organo genesis between differentiation of lens tissue from ectoderm and the formation of secondary lens fibers surrounding the embryonal lens nucleus. The exact time of the teratogenic insult and the morphogenesis of the abnormality were not determined in the study. The developmental lens abnormality appears to be unique because it has not been described as a compound-related abnormality-^. A similar-appearing structural lens abnormality has been reported to occur spontaneously in rat fetuses, but with a very low incidence of 1.2%^. The abnormality resembles the Fraser developmental lens abnormality of a mutant mouse strain which results from degenerative primary lens cells^. References 1. Coulombre AJ, Coulombre JL: Abnormal Organogenesis of the Eye, in Wilson J,, Fraser FC (eds): Handbook of Teratology:2 Mechanisms and Pathogenesis. New York, Plenum Press, 1977, pp 329-341. 2. Coulombre AJ: The Eye, in DeHaan RL, Ursprung H (eds): Organogenesis. New York, Holt Rinehart and Winston, 1965, pp 227-232. 3. Mann I: Development Abnormalities of the Eye, 2nd ed. Philadelphia, JB Lippincott Co., 1957. 4. Weisse I, Niggeschulze A, Stotzer H: Spontaneous congenital cataracts in rats, mice and rabbits. Archiv Fuer Toxikologie 32: pp 199-207, 1974. 5. Hamai Y, Kuwabara T: Early cytologic changes of Fraser cataract. An electron microscopic study. Investigative Ophthalmology 1_4_ (7): pp 517-527, 1975. 000620 Table 1 Oral Teratology Study of FC-95 in Rats Mean Maternal Body Weights with Standard Deviations 6 Dose Group 0 mg/kg/day 1 0 mg/kg/day 5 mg/kg/day 1 mg/kg/day Gestation Day 36 9 12 15 2 0 MEAN 200 "> "Ij- 247 "7 "*pj 2-80 STAN. DEV 16. 7 17. 6 20. 9 20. 5 24. 4 MEAN 199 22*2- 2`43- 257. 277 a 2 4 3 a STAN. DEV 11. 8 IS. 8 18. 2 16. 2 18. 6 24. 6 MEAN 20*;.' 228 249 268 294 S TAN. DEV 20. 0 16. 4 12. 6 12. 2 17. 8 22. 8 MEAN 05 226 252 272 202 3-78 STAN. DEV IS. 8 19. 1 19. 7 19. 5 24. 6 ~'i" * -- Significantly lower than the controls (Dunnett's t test p <0.05) 000621 Table 2 Oral Teratology Study of FC-95 in Rats Mean Litter Data and Pup Weights with Standard Deviations-- i Dose Group 0 mg/kg/day 10 mg/kg/day 5 mg/kg/day 1 mg/kg/day No. of Animals V I R B L E Ft FUSES M F TUI RL 20 5 . 2 4 . 9 1 0 . 0 1 . 2- 1 2. 17 --. O 9 (\ c cl.C' 2 . 6 4 . 3 17 3. 0 cr 1 0 . 5 1 . 9 "> 0 2 . 2 19 4 . 7 5. 4 1 0 . 1 i. ? 2 . 1 -i DE HD P.ESOEP TI Of- 1T1PLHH IR 11UH C O R P O P R M E H N MT. PE TU S ES sues SITES LUI EH F E T U S 03.) 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 1 0 . cl 0 . ,7 0. 9 0. 4 0. 6 0. ? 1. 0 0. 4 . 0. 8 10 . 8 2. 5 y. 1 4. 3 11. 2 22 10 . 6 2. ? 11. 2 i=l . f 92 3. 1 11. 1 2. 0 10. 9 2. 6 4. 3 0. 4 4. 3 0. 3 4. 2 0. 3 4. 2 0. 4 Treatment groups were not significantly different from controls (Dunnett's t test p < 0.05) 000622 o Table 3 Oral Teratology Study of FC-95 in Rats Number of Fetuses with Gross Findings'* Finding No. of fetuses examined Umbilical hernia Runted Total Normal Fetuses Total Abnormal Fetuses 0 mg/kg/day 201 1 -- 200 1 10 mg/kg/day 131 -- 1 130 1 5 mg/kg/day 178 1 -- 177 1 1 mg/kg/day 192 -- 1 191 1 -- Treatment groups were not significantly different from the control (Chi-square p <.0.05) 000623 9 Table 4 Oral Teratology Study of FC-95 in Rats Number and Percent of Fetuses with Skeleton Findings-- 0 10 5 1 Skeleton Finding_____________ mg/kg/day_____ mg/kg/day_____ mg/kg/day_____ mg/kg/day Fontanelle not closed 10 (7) Frontal nonossified 4 (3) Parietal nonossified 2 (1) Interparietal nonossified 3 (2) Occipital nonossified 1 (1) Stemebrae nonossified 114 (81) Stemebrae asymmetrical 53 (38) Stemebrae bipartite 7 (5) One stemebrae missing 30 (21) Two stemebrae missing 10 (7) 13 ribs 5 (4) 13 ribs spurred 7 (5) Wavy ribs 1 (1) Protrusion on ribs 6 (4) One body of the vertebrae 32 (23) bipartite Two bodies of the vertebrae 18 (13) bipartite Three bodies of the vertebrae 4 (3) bipartite Four bodies of the vertebrae __> bipartite 10 (ID 1 (1) 1 (1) ---- 1 (1) 77 (85) 23 (25) 4 (4) 13 (14) 2 (2) 2 (2) 7 (8) 2 (2) 9 (10) 21 (23) 7 (8) 1 (1) ,, --._ 7 (6) ---- 1 (1) ---- ---- 100 (81) 36 (29) 5 (4) 26 (21) 4 (3) 2 (2) 8 (7) ---- 3 (2) 25 (20) 11 (9) 1 (1) 5 (4) 1 (1) 1 (1) 1 (1) ---- 107 (81) 39 (29) 6 (5) 26 (20) 6 (5) 6 (5) 4 (3) 1 (1) 8 (6) 32 (24) 9 (7) 3 (4) 1 (1) Total No. Normal Fetuses 7 (5) 3 (3) 10 (8) 10 (8) Total No. Abnormal Fetuses 133 (95) 88 (97) 113 (92) 123 (92) Total No. of Fetuses Examined 140 91 123 133 -- Treatment groups were not significantly different from the control (Chi-square p < 0.05) ( ) = percent of total examined 000624 10 Table 5 Oral Teratology Study of FC-95 in Rats Number and Percent of Fetuses with Internal Findings Internal Finding 0 mg/kg/day Eye abnormality Thoracic cavity full of blood Enlarged atria Enlarged renal pelvis area in the kidney Abdominal cavity full of blood 1-- (2) -- 1 (2) 3 (5) 4 (7) Total No. Normal Fetuses 52 (85) Total No. Abnormal Fetuses 9 (15) Total No. of Fetuses Examinedl 61 10 mg/kg/day 14-- (35)-- 1 (3) -- -- 2 (5) 23 (57) 17 (43) 40 5 mg/kg/day 4-- (7) -- -- -- 5 (9) 47 (85) 8 (15) 55 1 mg/kg/day 2-- (3) -- -- 3 (5) 2 (3) 53 (90) 6 (10) 59 -- Eye abnormality was an artifact and was not considered for statistical b evaluations -- Eye abnormalities were developmental lens abnormalities with secondary lens aberrations -- Significantly higher than the control (Chi-square p < 0.05) ( ) = percent of total examined 000625 Appendix I Objective Oral Teratology Study of FC-95 in Rats Protocol A teratology study will be used to evaluate the embryotoxic and teratogenic effects of orally administered FC-95 to pregnant rats during the period of organogenesis. The procedure complies with the general recommendations of the FDA issued in January, 1966 ("Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use"). The study will be conducted according to the 1978 Good Laboratory Practice regulations and Safety Evalu ation Laboratory's Standard Operating Procedures. Sponsor 3M Commercial Chemical Division, St. Paul, Minnesota. Testing Facility Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. Study Director E. G. Gortner Start of Dosing Mid July, 1980. Test System Eighty-eight sexually mature, time mated Sprague-Dawley derived female rats from Charles River Breeding Laboratory will be housed in hanging stainless steel cages with wire mesh floors and fronts in a temperature and humidity controlled room. This strain of rats will be used because of historical control data and time mated females are readily available. Purina Laboratory Chow and water will be available ad libitum. The lights will be on a 12 hour light/dark cycle. Test System Identification Each animal will be ear tagged and that number will be indicated on the outside of the cage. 000626 Appendix I (Continued) 12 Randomization The animals will be assigned cages according to a computer-generated random numbers table. Control Article C o m oil. Test Article FC-95. Analytical Specifications The test article, composition and purity will be determined by the Sponsor (3M Commercial Chemical group) prior to the start of the study and at the end of dosing. Dosage Levels and Experiment Design The test article will be suspended in c o m oil daily. The test article suspension and control article will be administered by oral intubation to the rats on days 6 through 15 of gestation according to the following: Dose Group Dose Level Group Size High Mid Low Control 10 mg/kg/day 5 mg/kg/day 1 mg/kg/day 0 mg/kg/day 22 $ 22 9 22 9 22 9 The oral route of administration will be used because of metabolism studies showed radiolabeled FC-95 was well absorbed. No dietary contaminants are known to interfere with the test article. The animals will be observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights will be recorded on days 3, 6, 9, 12, 15 and 20 of pregnancy and the rats dosed accordingly using a constant dose volume of 5 ml/kg of body weight. The females will be killed on day 20 and the ovaries, uterus and its contents will be examined to determine: number of corpora lutea, number of fetuses (live and dead), number of resorption sites, number of implantation sites, pup weight and gross abnormalities. Approximately one-third of the pups will be fixed in Bouin's solution for subsequent free-hand sectioning by the Wilson technique to determine any visceral abnormalities using a dissecting 000627 Appendix I (Concluded) 13 microscope. The remaining approximately two-thirds of the pups will be fixed in ethyl alcohol for subsequent skeletal examination after clearing and staining with alizarin red. Data Analysis and Final Report The proposed statistical methods to be used for analysis of the data are: Dunnett's t test for dam and pup weights, number of fetuses, number of resorption sites, number of implantation sites and number of corpora lutea; Chi square for percent abnormalities. The proposed date for the final report is 2-3 months after detailed pup examinations have been completed (approximately fourth quarter, 1980). 000628 Appendix II Oral Teratology Study of FC-95 in Rats List of Principal Participating Personnel NAME Edwin G. Gortner Elden G. Lamprecht Cathy E. Ludemann Gary C. Pecore Loren 0. Wiseth FUNCTION Study Director Veterinary Pathologist Coordinator-Histology Supervisor-Animal Care Technician 000629 Appendix III STATEMENT OF QUALITY ASSURANCE STUDY NUMBER: TITLE: Q680TR0008 Oral Teratology Studv of FC-95 in Rats Audits and/or inspections were performed by the Riker Q u a lity Assurance U n it f o r th e above t i t l e d study, and rep orte d to the stu dy d ir e c to r and to management as fo llo w s : Date Performed 18 July 1980 28 July 1980 15 December 1980 17 December 1980 Date Reported 21 Julv 1980 28 July 1980 17 December 1980 17 December 1980 1. E. Orterstrom Laboratory Q u a lity Assurance Riker Laboratories, Inc. December 17, 1980 Date 000630 APPENDIX IV 16 Test and/or Control Article Characterization for _____ F C - 9 5 . L M 6 M O __________ 1. The identity strength, uniformity, composition, purity or other per tinent characterizations of the test and/or control substances have been determined and documented as of /hfty ft", /d'O____________ . 2. The method of synthesis or origin of the test and control substances, including their amount and the method of bioassay (if applicable) is documented. /. yes A no _____ 3. The stability of the test and/or control substances have been deter mined or will be determined as of d c 6f The above information and documentation are located in the sponsor's re cords . %-l-JL. Sponsor iU Date 000631 PWO Appendix V Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group _____________ Study Day____________ and Rat No. 3 6 9 12 15 20 0 t'lG/KG/DttV NOR 12396 NGR 1293? N0R 12338 N0R 12339 NGR 13000 N0R 13016 NGR 0 0 1 8 NGR 13013 N0k 13020 NQR 13036 NGR 13040 N0R 13041 NGR 13043 N0R 13044 N0R 13060 N0R 13061 N0R 13062 N0R 13063 N0R 13064 N0R 13080 186 212 224 261 216 2!2o 212 222! 224 250 182 207 175 201 193 219 194 221 208 22!y 186 208 133 21.9 220 2!29 20 r:' 228 226 248 190 212 210 2!2!S' 180 208 188 211 179 134 235 22$# 240 271 240 284 203 22 r*' 3;6 2!*22 222 253 284 235 213 222 24 7 2~i5r.|013. 254 306 *zLii 274 307 205 246 2/7 c.rr' 284 261 258 290 2 *'2 310 259 2 *,2 25 i*' 256 238 cLc*r 345 315 302 335 284 cLi' 309 319 222 209 340 296 343 297 202 2S9 W 25G 25 7 424 39 7 --l'*--* 435 24 2 804 2*iC1 400 408 381. 350 426 359 442 366 262 342 268 221. HERN 200 2!2!' 24? 272 305 280 STRN. DEV lit*. r'` 17. 6 0. 9 20. b 24. 4 NON PEEGNHN 1 RNI UHLS N0R 13017 186 198 242 217 234 252 N0R 13042 188 209 2601 247 255 272 000632 17 Appendix V (Continued) Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group and Rat No. 3 Study Day 6 9 12 15 20 10 MG/KG/C>HV O0R 12001 D0R 13002 G0R 12:002U0R 12004 O0R 12:005 0R 13021 00R 12022 00R 12022 00R 13025 U0R 13022 O0R 12045 00R 12040 00R 13065 O0R 13066 00R 12:067' 00R 13069 00R 130S1 162 190 192 192 201 idid7 212 180 208 187' 195 186 204 20 7' 210 203 194 222 217' 222 212 c.-.id c_r* 257 244 206 22 " 214 216 285' 223 226 224 22y 209 23S* 220 224 218 260 24 7 243 258 251 22'y 226 226 274 275 222 262 228 256 265 232 261 2 7S 2yy 227 268 250 259 236262 262 268 262 237 281 ,,*>O 290 255 292 211 245 297 274 269 248 279 26*2 278 283 251 3 4y 306231 219 26-9 360 402 285 3od 257 261 311 304 3-2'2 33o 281 MERN 199 242 257 d ('r' 243 STRN. DEV 11. 8 13. 8 18. 2 3.6. 2 18. 6 24. 6 n u n PREGNHN1 RNIMRLS O0R 12024 195 217 222 22:0 242 252 O0R 13046 187 209 242 228 2j:2 231 00R 13047 184 201 244 221 233 235 00R 13049 212 2 3 y 242 250 251 266 00R 12068 216 23-2' 236' 239 25 261 000633 18 ia >r>. BESTCOPYAVAILABLE Appendix V (Continued) Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group and Rat No. 3 Study Day 6 9 1 2 15 2 0 5 MG/'KG/DPIV 6 6 0 1 -1 -0 0 6 PUP 1 jUU7 r'ijK 1 2 U U 8 PUP 12069 PUP 1201U PUP 1262 7 PUP 12028 PUP. I G U ^ P pup: iuuuu PUP: l i U i o 1_2 0 60 PuP: 1 2 U 5 1 PUP 13U52 PUP H U 5 4 PUP 12U7U P U P 12U.-1 PUP 12072 132 226 197 188 194 2 12 2 12 199 17t. 198 18 8 222 222 197 224 20U 188 2 18 243 222 2 12 220 2 ji-2 2 ---* 2 2 !t* 2 1U 219 2U4 242 248 224 2 t*6 222 211 |S1'-2' 272 262 274 248 251 2 --0 241 260 225 2 2 ^* 2 bT.* 242 2 -i-;i: 245 25U 26U 258 264 262 254 26-p265* 274 d r`d. 27U 262 246 262 291 259 237 274 256 272 204 200 2 0 -i 282 200 294 200 294 20 0 260 S2 2 - 225 279 d' 204 232 24U 200 294 261 242 263 S0 -! 2 6*6 vQ 26 6 J'2 4U7 4U8 243 41U --l''0 262 HERN 2U2 228 249 260 294 8 TMl2 L-'EV 20. 0 16 4 12. 6 12. 2 17. 8 2S *z* NUN PPEGNHNT HNif'lHLS PUP 12U26 217 22 ij 294 252 252 261 PUP llU5'2 218 2 S 252 248 254 262 PUP 12672 206 221 250 250 244 2,c;3 PuP: 12 174 20 7 224 244 ~icv 272 28'7. P0P 12-U82 195 214 240 225 222 240 000634 19 KST COPYAVAILARif Appendix V (Concluded) Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) 20 Dose Group and Rat No. 3 Study Day 6 9 1 2 15 2 0 1 MG/KG/Dft V Q0k 13-011 OtR 12012 QOk 12012 GlOR 12014 GlOR 13-010 U0k 12021 Q0R 12022 WOk 12032 G-!0k 1.3:03.4 U0k 12030 G.!0k 13628 Q0k 12000 Q0k 12006 00k 1200? G`0R 1200' 8 Q0k 120701 Q0R 12077 G!0R 12078 Q0R 13:082 188 21? 182 188 2 Gi0 224 180 204 182 180 225 201 204 188 201 180 188 208 261 224 220 204 221 228 2018 220 220 2 2' 201 220 22S 211 224 206 210 228 278 200 248 23:0 224 241 246244 204 2 2 202 2S2 2 cj 9 2 2 6> 264 28-226.3 242 276 261 282 26-7' 2 r*2 284 20*j 200 26*9 262 201 201 261 26-2 250 27 0 257 262 26.2 222 288 210 200 201 3:26* J.22 2 76 320 286* 271 224 302 286 26-8 301 281 296 297:` 36*`9 3-6*7 286* 379 376* 413 411 3-22 407 200 J;..cr"> 416 278 271 3-3 32 70 362 3 63 374 408 MEh N 200 226 202 ci r`2 202 278 STHN. DEV 18. 8 18. 1 19. 7 18. 0 24. 6* 21. 8 N O N RREGNHf'.i R N I M R L S Q0 R 12008 G.!0k 1 2 0 7 6 G!tiR 1 2 0 7 8 183 182 186 200 2 12 218 24 F 226* 228 2 2 ^* 2 204 278 244 200 t-|-r- 270 2 66 000635 B K r C 0PK4W ft4ltt Appendix VI Oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights Dose Group and Rat No VIABLE FE TUSES DEAD EESUE IMPLAN CURF'RH MEHN FETUS R7 >,G> M F TOTAL FETUSES PTION TATIUN LUTEH AVG M F SITES SITES 0 mg/kg/day N0 R N0R N0R N0R N0R N0R N0R N0R N0R N0R H0R N0R N0R N0R N0R N0 R NOR NOR N0R N0 R NOR N0R 12996 12997 12998 12999 13000 13016 1301? 13018 13019 13020 13036 13040 13041 13042 13043 13044 13060 13061 13062 13063 13064 13:080 54 Cj 4 13 *' 4 1 1 r* 4 11 ' r* 14 4u Cj NUT PREGNANT i' 3 10 61 r 48 IT 5 12 10 6 f* 13 6 3 NUT PREGNANT 46 5 10 7 8 5 13 46 10 4 r' 15 5 r* 1 2 s2 s MEAN 5. 2 4. 9 1 0 . 0 Ta n . d e v . 1. 7 2 . 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .0 0 .0 1 10 1 14 0 11 '1 13 0 14 1 10 1 11 0 r" 0 12 1 11 0 13 1 10 13 0 1* 1 14 2 12 0 i' 0 t' 0 12 19 0. 7 1 0 . 8 0. 9 2 . 5 9 16 12 13 17 9 ii s 12 8 12 12 15 11 12 11 9 9 12 9 1 1 .2 4. Cl J:-. 4. s 4. 0 4. 1 Ji-. r' 5. 1 s.y 4. 3 4. 1 4. 2 Ji-. 4. 7 3-. 5 4. 2 . 9 4. 0 4. 0 4. if, 5. l 4. r' 4. i 4. 4 4. i=: 4. 5 5. 1 4. 8 4. 2 4. 5 4. 4. 3 4. 9 4. 7 9 4. 2 4. 1 42 S. q 4. i 4. cL 4. 1 4. s 4. cL 4. 4 4. 4 . O 4. 1 4. 3 4. 4 4. 4. 2 4. 4 4. 1 U 2. 3 4. 1 . y 4. 3 4. 2 4. 2 4. 0 .4 000636 22 Appendix VI (Continued) Oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights and Rat No VIABLE FETUSES DEAD RESOR IARLAN CORRRA MEAN FE'IUS HI <.ia> M F TOTAL FETUSES FT ION TAT ION LUTEA AVG H F SITES SITES 10 m g A g / d ay OOF: 13001 00k 13002 OOF': 13003: O0R 13004 O0R 13:005 O0R 13021 O0R 13022 U0R 13023 O0R 13024 O0R 13025 O0R 13037 O0R 13045 00k 13046 OUR 13047 00 R 1304S' OUR 1304S O0R 13065 O0R 13066 O0R 13067 U0R 13068 O0R 13069 O0R 13081 4 6 10 3* 6 3 4 7 ii f' d. 3 5 7 12 13 4 11 2 13 20 2! NOT PREGNANT 6 b 12 5 5 10 4 6 10 NOT PREGNAN1 NOT PREGNANT 5 3: s NOT PREGNANT 01 1 4 8 12 11 ci! NOT PREGNANT ScL d._"i 01 i MEAN 3. 8 3. 3 *'. c TAN. DEV. 2!. S 3. t* 4. 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0. 0 0. 0 1 11 i! 11 0 11 03 0 12 04 0 13 02 0 12 0 10 1 11 13 0i 0 12 1 1 fc, 01 0. 4 8. 1 0. 6 4. 3 10 11 12 3 12 r* 14 5 12 12 11 s 6 ii 8 3. 2 2. 1 4. 5 4. 6 4. b *?. 4. 1 s. y 4. 2 4. 4 4. i 4. 2 4. 2 4. G 4. 3 4. 3 4. 2 4. 4 4. 4 4. 4 4. 2 4. 2 -i, y 4. y 4. 8 0. 0 4. 4 4. 5 4. 4 4. 3 4. 4 4. 4, 5 4. 6 4. 4 4. 2 4. 3 4. 3 0. 0 4. 0 4. 2 4. t- 4. 8 4 Jl- J*.. 3 4. 5 S . jl- s . *' 4. 1 0. 0 4. 1 4. 3 0. 3 000637 BEST copy AVAILABLE Appendix VI (Continued) Oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights 23 Dose Group and Rat No. V1HBLE FETUSES DEFID RESOR IMPLRN CQRPRR HERN FETUS WI G -> M F TOTHL FETUSES FT ION lHTION LUTER RVG M F SITES SITES 5 mg/kg/day F'OF: IS OOt. F'UF: ISOG 7 F'OF, 12U08 F'OF! ISOOS F'OF! 12010 POF: 12088 F'UF: 12027 F'OF! 12028 FOR 1202S F'OF: 1202:0 F'OF: 12028 F'oF: 12050 F'OF: 12051 FOF: 12052 FOR 12052 FOR 12054 FOR 12070 FOR 12071 FOR 12072 FOR 12072 FOR 12074 FOR 12082 -S . `9 12 12 54 9 4 l' ii NOT FREGNHN 1 8 ii 4 !"! 12 4 j-. r* 4 9 12 5 5 10 4- 5 3 4 f" ii NO 1 FREONHN1 Cl 14 4 10 5 8 12 S9 4CT. NOT FREGNRNT NOT FREGNRNT NOT FREGNRNT MERN 5. 0 5. 5 10. 5 s TRN. DEV 1. S 2. O 0 O O 0 O 0 0 0 0 0 6 O 0 0 O 0 0. O HT 0 9 9 4. 8 4. 8 4. 4 O 12 12 4. 2 4. 4 4. 0 0 12 12 "1; 9 4, s. 8 09 o S.O 4. 1 -i-. ts 8 ii 12 3. 3 4. 0 y i 12 10 4. O 4. 1 8_s. 0 12 12 4. 2 4. 4 4. --- 8 10 4. 8 0. 2 4. 1 14 14 4. 5 4. 5 4. --1 0 10 10 4. 4 4. 7 4. 2 1 10 9 4. O 4. 2 9 _s. k! 12 12 4. 2 4. 4 4. 2 O 14 14 3. 6 S. r 5 0 10 11 4. 2 4. 2 4. 1 2! 15 14 4. 2 4. 2 4. 2 i 10 9 4. 4 4. 7 4. J;- 09 9 4. 2 4 4 4 2 0. 7 11. 2 ii. i 1. 0 ci.CL 2. O 4. 2 0. 3 000638 24 BESTCOPYAVAILABLE Appendix VI (Concluded) Oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights Dose Group and Rat No. V I A B L E F E T U S E S DEAL' ri p T O T A L F E T U S E S RESOR IMPLAN CORPRH FT ION TAT ION L U T E A SITES SITES MEAN PETU AVG M 1.G . 1 mg/kg/day OOP 13011 OOP 13012 OOP 130:12 G!Ok 12014 OOP 12010 OOP 120ol OOP 12022 OOP 12023 OOP 13034 OOP: 13:03:5 OOP: 13U3S OOP 13:0 5 OOP 13:056 OOP 13057 OOP 13:058 OOP 1205S QOR 12075 OOP: 12076 OOP 13077 OOR 13078 OOP 1307S OOP: 13083 i* 10 0 6 11. J;. b Q 5 i'" 12 4 b 10 i' b 1.2 1i cL 4 Q 13 cl' 4 b 5 5 lo b fc. 12 j? 4 11 5 fc. 11 45 3 NOT PREGNAN1 6 4 10 b 4 10 NOT PREGNANT C=, 5 5 10 NOT PREGNANT 4 11 15 MEAN 4. 7 5. 4 10. 1 ST AN. DEV. 1. 7 2. 1 0 0 O 0 O O 0 0 0 0 0 0 i 0 0 0 0 0 0 0. 1 O. 2 0 10 0 11. 0 Cj O 12 0 10 0 12 03 0 13 J- 3 1 ii 0 12 1 12 1 13 1 10 0 10 0 10 13 0 10 0 15 0. 4 10. 6 0. 8 ft 12 11 13 3 14 4 14 8 11 12 12 11 12 11 11 3 10 15 10. 9 b2 .. 4. 4 4. 1 4. 4 3. 4 S. b 4. 0 -ii-. I"i 4. 5 5. 0 4. 6 4. 2 4. 2 4. 1 3. 9 4. C4. 1 44 .8 8 Jj..3 4. 4. b i. J t m 4. 4. 4 4. si! 4. 2 2. 8 4. 4 4. 0 4. 2 .2 3. 8 4. 1 --. 4. 5 4. S 4. 4 4. 2 4. 3 4. 0 39 4. 1 4. - 3. 8 4. 2 4. 4. 1 4. 4 4. 4 4. 4 4. 6 4. ft 4. 4 4. 1 4. 4. O 4. 2 O. 4 000639 DISTRIBUTION LIST K. L. Ebbens -* J. D. Henderson G. R. Steffen M T. Case E. G. Gortner (original +1) F. Keller (QA file) E. G. Lamprecht R. A. Nelson - E. L. Mutsch R. E. Ober 000640 BEST COPY AVAILABLE cc: M. T. Case E. L. Mutsch -* H A. Nelson -* R. E. Ober E. G. Gortner (original + 1} F. Keller (QA File) PearIson E. G. Lamprecht G. R. Steffen J. D. Henderson K. L. Ebbens Amendment to the Final Report of the Oral Teratology Study of FC-95 in Rats Experiment No.: 0680TR0008 Issued: 12/18/80 Please add the amended summary, the amended table 5, and the amendment to the results and discussion sections to the above report. The study conclusions were changed by this amendment to the report. E. G. Gortner Date Senior Research Technologist Animal Teratology Reproduction E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist 9)2(5UAfM^ 7 SUL 7j27/?J2- M. T. Case, DVM, PhD ^ Date Manager, Pathology-Toxicology Safety Evaluation Laboratory 000641 BEST COFY AVAILABLE Amended Summary (p. 1) to the Oral Teratology Study of FC-95 in Rats Experiment No. 0680TR0008 Oral administration of FC-95 at doses of 10, 5 and 1 mg/kg/day to pregnant Sprague-Dawley' rats during days 6 through 15 of gestation (period of organogenesis) was not teratogenic. FC-95 administration was maternally toxic only to the 10 mg/kg/day group. At gestation days 12 through 20 the maternal body weights of the high dose females were significantly lower than the controls. FC-95 was not maternally toxic to the 5 and 1 mg/kg/day groups. FC-95 was not embryotoxic and did not affect the ovaries or reproductive tract contents of the dams. The compound did not produce an increase in the number or proportion of fetal skeleton variations. 000642 Amendment to the Results and Discussion Sections (p.3-5) of the Oral Teratology Study of FC-95 in Rats Experiment No. 0680TR0008 (This amendment addresses the last two paragraphs of the results section and the entire discussion section.) FC-95 was labeled a teratogen of the lens because apparent lens abnormalities were observed at the 10, 5 and 1 mg/kg/day dose levels. Based on subsequent studies, particularly Riker Experiment No. 0681TR0362, the interpretations of these observations have been extensively modified. The lens findings observed under the dissecting microscope are now known to be either freehand sectioning artifacts or a normal area of lens cell degeneration. The fetal rat lens findings were incorrectly interpreted as a teratogenic change in this study. The gross finding of a lens cleft was an artifact created by freehand sectioning. It represents a separation between the embryonal nucleus lens cells and the lens epithelium. The gross finding of a lens dark streak was a normal observation of the embryonal nucleus. The embryonal nucleus is an area of normal lens cell degeneration in the gestation day 20 fetus. The gross appearance of the rat lens at day 20 of gestation is determined by the region of the lens which is transected by freehand sectioning. In a subsequent study (Riker Experiment No. 0681TR0362) the compound-related occurence of the lens findings could not be repeated when the fetuses were coded before freehand sectioning and gross evaluation. The range of gross lens observations and the differences among the dose group incidences were due to the manner and frequency in which the lens cleft artifact was created by freehand sectioning and the limitations inherent in visualizing the embryonal nucleus. In summary, FC-95 in utero exposed fetuses did not have compound-related changes in their lenses. 000643 Amended Table 5 (p. 10) Oral Teratology Study of FC-95 in Rats Number and Percent of Fetuses with Internal Findings 0 10 51 Internal Finding____________ m g A q / d a y _____ mg/kg/day______ mg/kg/day_____ mg/kg/day Lens findings Thoracic cavity full of blood Enlarged atria Enlarged renal pelvis Abdominal cavity full of blood 1 (2) 1 (2 ) 3 (5) 4 (7) 14 (35)*i 1 (3) 2 (5) 4 (7) 5 (9) 2 (3) 3 (5) 2 (3) No. of Fetuses Examined 61 40 55 59 -- The lens findings observed under the dissecting microscope were either freehand sectioning artifacts or a normal area of lens cell degeneration -- Significantly higher than the control (chi-square with Yates correction p < 0.05) ( ) = percent of total examined 000644 DISTRIBUTION LIST K. L. Ebbens E. G. Gortner (original + 1) F. D. Griffith -* F. A. Ubel F. Keller (A File) E. G. Lamprecht R. E. Ober R. A. Nelson -* E. L. Mutsch W. H. PearIson G. R. Steffen -* J. D. Henderson + M. T. Case W. C. McCormick 000645 M M COPY AVAILABLEAmended Appendix VII STATEMENT OF QUALITY ASSURANCE STUDY NUMBER: Amendment to 0680TR0008______ TITLE: Amendment to the Final Report of the Oral Teratology Study of FC-yb in Rats Audits and/or inspections were performed by the Riker Compliance Audit unit for the above titled study, and reported to the study director and to management as follows: Date Performed Date Reported July 16 and 19, 1982 July 22, 1982 July 21, 1982 July 23, 1982 Comp iahde a u dit Riker Laboratories, Inc. Date 000646 BEST COPli AVAILABLE Kr DISTRIBUTION LIST K. L. Ebbens -* J. D. Henderson + G. R. Steffen -* M. T. Case E. G. Gortner (original +1) F. Keller (QA file) E. G. Lamprecht R. A. Nelson - E. L. Mutsch R. E. Ober 000647